The present invention relates to nucleic acid constructs, pharmaceutical compositions and methods of using such constructs for treating cancer.
Neoplasia is a process that occurs in cancer, by which the normal controlling mechanisms that regulate cell growth and differentiation are impaired, resulting in progressive growth. This impairment of control mechanisms allows a tumor to enlarge and occupy spaces in vital areas of the body. If the tumor invades surrounding tissue and is transported to distant sites (metastases) it will likely result in death of the individual.
The desired goal of cancer therapy is to kill cancer cells preferentially, without having a deleterious effect on normal cells. Several methods have been used in an attempt to reach this goal, including surgery, radiation therapy and chemotherapy.
Surgery was the first cancer treatment available, and still plays a major role in diagnosis, staging, and treatment of cancer, and may be the primary treatment for early cancers. However, although surgery may be an effective way to cure tumors confined to a particular site, these tumors may not be curable by resection due to micrometastatic disease outside the tumor field. Any cancer showing a level of metastasis effectively cannot be cured through surgery alone.
Radiation therapy is another local (nonsystemic) form of treatment used for the control of localized cancers. Many normal cells have a higher capacity for intercellular repair than neoplastic cells, rendering them less sensitive to radiation damage. Radiation therapy relies on this difference between neoplastic and normal cells in susceptibility to damage by radiation, and the ability of normal organs to continue to function well if they are only segmentally damaged. Thus, the success of radiation therapy depends upon the sensitivity of tissue surrounding the tumor to radiation therapy. Radiation therapy is associated with side effects that depend in part upon the site of administration, and include fatigue, local skin reactions, nausea and vomiting. In addition, radiation therapy is mutagenic, carcinogenic and teratogenic, and may place the patient at risk of developing secondary tumors.
Other types of local therapy have been explored, including local hyperthermia, photoradiation therapy and interstitial radiation. Unfortunately, these approaches have been met with only moderate success.
Local treatments, such as radiation therapy and surgery, offer a way of reducing the tumor mass in regions of the body that is accessible through surgical techniques or high doses of radiation therapy. However, more effective local therapies with fewer side effects are needed. Moreover, these treatments are not applicable to the destruction of widely disseminated or circulating tumor cells eventually found in most cancer patients. To combat the spread of tumor cells, systemic therapies are used.
One such systemic treatment is chemotherapy. Chemotherapy is the main treatment for disseminated, malignant cancers. However, chemotherapeutic agents are limited in their effectiveness for treating many cancer types, including many common solid tumors. This failure is in part due to the intrinsic or acquired drug resistance of many tumor cells. Another drawback to the use of chemotherapeutic agents is their severe side effects. These include bone marrow suppression, nausea, vomiting, hair loss, and ulcerations in the mouth. Clearly, new approaches are needed to enhance the efficiency with which a chemotherapeutic agent can kill malignant tumor cells, while at the same time avoiding systemic toxicity.
Because it is unlikely that gene transfer reaches every cell of a cancer, DNA based therapy approaches are thought to require the induction of a ‘bystander’ effect. An interesting and novel approach for this purpose is cytokine DNA based therapy. In particular, use of TNF-α seems an attractive strategy to prevent for example, bladder tumor recurrence after transurethral resection.
TNF-α is a multifunctional and immunoregulatory cytokine that exhibits direct tumor cell cytotoxicity, possesses antiangiogenetic properties, and enhances antitumor immunity by activating immune cells such as dendritic cells and T cells. Induction of TNF-α is thought to be partly responsible for the effect of BCG immunotherapy in the prevention of TCC recurrences (1,2) and recombinant cytokine therapy has—in principle—proved efficacious for bladder cancer (3). However, systemic delivery of the TNF-alpha protein has had limited success clinically because of severe dose limiting toxic effects.
This limitation can be overcome by the use of a gene delivery approach, combined with a tumor specific promoter to express TNF-α in the tumor tissue, optionally together with a tumor specific expression of a toxin.
H19 was the first human imprinted non protein-coding gene to be identified showing expression of only the maternal allele (Rachmilewitz et al, 1992; Zhang and Tycko, 1992). It is also imprinted in mice (Bartolomei et al, 1991). H19 was mapped on the short arm of chromosome 11, band 15.5, homologous to a region of murine chromosome 7 (Leibovitch et al, 1991). It belongs to a group of genes that very likely does not code for a protein product (Brannan et al, 1990).
Studies of various tumors have demonstrated a re-expression or an over-expression of the H19 gene when compared to healthy tissues. Moreover in cancers of different etiologies and lineages, aberrant expression in allelic pattern was observed in some cases. While H19 shows mono-allelic expression in most tissues throughout development, with the exception of germ cells at certain stages of maturation, and in extra-villous trophoblasts, bi-allelic expression of this gene, referred as “relaxation of imprinting” or LOI, have been found in an increasing number of cancers, for example, hepatocellular carcinoma, liver neoplasms of albumin SV40 T antigen-transgenic rats, lung adenocarcinoma, esophageal, ovarian, rhabdomyosarcoma, cervical, bladder, head and neck squamous cell carcinoma, colorectal, uterus and in testicular germ cell tumors. Today nearly 30 types of cancers show dysregulated expression of H19 gene as compared to healthy tissues, with or without LOI. For recent review see (Matouk et al, 2005).
It was also shown that H19 over-expression of ectopic origin conferred a proliferative advantage for breast epithelial cells in a soft agar assay and in several combined immunodeficient (SCID) mice (Lottin et al, 2002). In tumors formed by the injection of cells of a choriocarcinoma-derived cell line (JEG-3), and a bladder carcinoma cell line (T24P), the H19 level is very high when compared to the level of H19 in cells before injection (Rachmilewitz et al, 1995; Elkin et al, 1995; Lustig-Yariv et al, 1997).
Moreover, certain known carcinogens upregulate the expression of the H19 gene. A dramatic elevation of H19 RNA levels was detected in the airway epithelium of smokers without loss of imprinting (LOI) (Kaplan et al, 2003). BBN (N-butyl-N-(4-hydroxybutyl nitrosamine, a known carcinogen of the bladder) also induces the expression of H19 gene in the rat model of bladder cancer (Elkin et al, 1998; Ariel et al, 2004). Likewise, Diethylnitrosamine (a known carcinogen of the liver) induces the expression of H19 in a mice model of hepatocellular carcinoma (Graveel et al, 2001).
The specific expression of H19 gene in cancer cells has prompted its use in clinical applications for diagnosing cancer.
Thus, U.S. Pat. No. 5,955,273 to the present inventors teaches the use of H19 gene as a tumor specific marker.
PCT Pub. No. WO 9524503 teaches the detection of malignancies and their grading with a H19 gene probe by in-situ hybridization—useful for detecting presence/absence of malignancy in pediatric Wilms' Tumor.
PCT Pub. No. WO 04024957 teaches detecting cancer or the presence of residual cancer cells or micro-metastasis by detecting the presence of H119 RNA in the specimen.
The use of H19 promoter for specifically expressing cytotoxic agents in cancer cells has been suggested in PCT Pub. No. WO9918195 which teaches the specific expression of heterologous sequences, particularly genes encoding cytotoxic products, in tumor cells under the control of regulatory transcriptional sequences (e.g., H19 promoter).
To date cancer-specific gene therapy using TNFα and diphtheria toxin A under a cancer specific promoter has never been suggested or attempted.